A Litografía

KORNÉL KOVÁCS

Biomass stores the energy captured from sunlight during photosynthesis in the form of chemical bonds holding the molecules together. In the various ways of producing renewable biofuels, this chemical energy is released and converted into a form that is easy to utilize in everyday energy consuming devices, e.g. transportation vehicles, light bulbs etc. The most commonly used fractions of biomass are the ones prepared for energy storage purposes by the photosynthe- tic organisms themselves. These are sugars or their polymers, such as starch or cellulose.

The transformation of starch into sugar is an important branch of the starch industry and one of the most important applications of biotechnology. Countless foods contain ingredients produced by the breakdown of starch. Enzymes are the key to these chemical reactions – enzymes that are predominantly produced with the help of genetically modified microorganisms. Starches are chemically bound clusters of sugar molecules found in plants. Under the right conditions, starch molecules can be broken down into sugar. Sugar is the most preferred carbon and energy source for almost all microbes used in biotechnological processes. This process makes it possible to obtain sugar from the starch of many different plants, rather than just sugar beets or sugar cane. This is now being done by industrial-scale starch saccharification. The most important sources of starch are maize, potatoes, and wheat.

Strong acids were once used to break apart starch molecules and release sugar. Now, enzymes do the job offering many advantages: with enzymes, the process targets the proper chemical bonds much more precisely. Different enzymes can be used to produce syrups with different levels of sweetness and different technical characteristics. The end products are not only used as custom tailored ingredients in countless foods and drinks, they can also be further processed into glucose, artificial sweeteners, or fat substitutes.

For a long time, enzymatic breakdown of starch (saccharification) did not make economic sense. Things changed, however, as soon as the enzymes responsible for this process became available at low cost, high quality, and in practically unlimited quantities. Now, almost all of the enzymes used to break down starch are produced with the help of genetically modified microorganisms.

Plants are used as a starch source. A certain portion of the raw material may be genetically modified. Cultivars of maize and potato have been produced, in which the structural properties of the starch molecules are altered so that saccharification takes place more efficiently. These starch sources are then used in bioenergy production, making subsequent fermentation of the sugar component more efficient and the overall process economically more viable. Next generation renewable energy carriers will utilize lignocellulosic raw material. Lignocellulose

is the material produced in the largest amount on Earth. However, due to its structural complexity direct utilization of this biomass by microbes is extremely low. Ongoing research aims at the production of efficient enzymes in large quantities that would make the utilization of lignocelluloses for energy production purposes economically feasible. These so called third generation energy carriers made from lignocelluloses, like ethanol, butanol or biomethane, biohydrogen, will make possible to stop the mass utilization of fossil fuels, which poison our environment and are rapidly depleted.

The production of energy is achieved via direct utilization of solar energy by phototrophic organisms or through dark fermentative conversion of biomass to valuable energy carriers. Photosynthetic microbes such as algae, cyanobacteria and phototrophic bacteria can be genetically modified so that their energy and/ or metabolic pathways are directed towards the production of useful molecules. A recent example for this strategy is the production of isobutyraldehyde by a genetically modified cyanobacterium. Cyanobacteria and algae carry out photosynthesis and using this energy source they build up their molecules from carbon dioxide, the major contributor to global warming and associated environmental problems. The genes of four enzymes, obtained from other bacteria, were incorporated into the genome of Synechococcus elongatus, a thoroughly studied cyanobacterium. As a result, a new biosynthetic pathway was engineered in the host cyanobacterium, which produces isobutyraldehyde from the CO₂ fixed. The product is volatile, thus its removal from the cyanobacterial culture is fairly easy. This compound is the precursor of several useful chemicals, including isobutanol, which has a great potential as fuel alternative to gasoline.

Among the alternative energy carriers hydrogen appears to be the most promising, because it burns to environmentally friendly water when utilized, and may be transported and stored rather easily. Hydrogen can be produced in biological processes: in algae and cyanobacteria solar energy captured by the photosynthetic apparatus is converted into chemical energy through water splitting, the reaction that yields oxygen and can also produce hydrogen. Upon utilization, these components are combined to form water and energy is released in a cycle driven by the Sun, a practically unlimited and safe energy source.

The understanding of molecular fundamentals of hydrogen production and utilization in microbes is a goal of supreme importance both for basic and applied research applications. The key enzyme in biological hydrogen metabolism is hydrogenase, which catalyses the formation or decomposition of the simplest molecule occurring in biology: molecular hydrogen. The simple-looking task is solved by a sophisticated molecular mechanism. Hydrogenase is a metalloenzyme, harbouring Fe or sometimes Ni and Fe atoms. Like most metalloenzymes, hydrogenases are extremely sensitive to inactivation by oxygen, high temperature and other environmental factors. These properties are not favourable for several potential biotechnological applications. In metal-containing biological catalysts it is the protein matrix surrounding the metal centres that provides the unique environment for the Fe and Ni atoms which allows hydrogenases to function properly, selectively and effectively. Therefore, a major goal of hydrogenase ba-

Kornél Kovács

sic research is to understand the protein-metal interaction. The problem is not simple to address, as some of the methods for scientific investigation provide information on the metal atoms themselves without directly observing the protein matrix around them. Other modern techniques at our disposal reveal details of the protein core, but do not display the metal centres within. A combination of the various molecular approaches is expected to uncover the fine molecular details of the catalytic action of metalloenzymes. Engineering the protein matrix by random and site-directed mutagenesis, expression of the enzymes in various hosts lead to genetically modified stable hydrogen producers. In addition, through metabolic engineering, i.e. switching genes the protein products of which take part in various energy production or consumption pathways, allow the guidance of energy towards hydrogen production (Figure 12.1).

Figure 12.1. Hydrogen production by a phototrophic bacterium

(Thiocapsa roseopersicina). The blue columns indicate the daily hydrogen production of the wild-type strain. A GM mutant (red columns) is capable to produce more hydrogen for a longer period of time using an altered metabolic route

Dark fermentation of biomass usually involves anaerobic biotechnology due to its potential to produce value-added products from low-value feedstock such as waste streams. In addition, it provides an opportunity for the removal of pollutants from liquid and solid waste more economically than other processes. Genetically altered metabolic routes give improved yields in the production of

600 s) Nitrogenase Hox1+ 400 500 ce d (G C un its 200 300 dr og en p ro du c 0 100 5 6 7 8 9 12 13 14 15 16 19 20 21 Tot al h yd 5 6 7 8 9 12 13 14 15 16 19 20 21 Days

biohydrogen, biomethane, biobutanol, bioethanol and biodiesel in anaerobic conversion of biomass to useful energy carriers. A rapidly developing area is butanol production. Certain Clostridia are capable of butanol synthesis during the so-called solventogenesis stage of their growth. During solventogenesis these bacteria predominantly produce a mixture of butanol, ethanol, acetone and some organic acids. In order to make the bacteria focus on butanol production, the numerous alternative pathways present in Clostridia should be blocked by knocking out the genes coding for some key enzymes in those pathways.

In the field of food production and preservation another group of anaerobic bacteria, the lactic acid bacteria (LAB) are used widely. Among other foodstuffs, cheese is made by them. Cheese is made from the protein-rich fraction of the milk. The main protein component in milk is casein, a hydrophobic protein stabilized in the aqueous environment by another protein called Kappa-casein. In order to recover casein from the milk, Kappa-casein should be removed, and this job is done by an enzyme named rennet or chymosin. Rennet has been traditionally obtained from the stomach of calves: it degrades proteins and thus upon addition to the milk it decomposes Kappa-casein leaving the hydrophobic casein behind, which precipitates and forms the curd. As large scale cheese production emerged in the 1960s, there was a shortage of rennet. Microorganisms have been modified genetically to yield chymosin that is identical to the enzyme obtained from animals. This can be used to produce better quality cheese than the original rennet. The first scientists to make chymosin in this way in 1981 used bacteria; chymosin is now obtained from yeasts. In 1988, chymosin was the first enzyme from a genetically-modified source to gain approval for use in food industry. These proteins behave in exactly the same way as calf chymosin, but their activity is more predictable and they have fewer impurities. Today about 90% of the cheese industry products are made using chymosin from genetically-modified microbes. It was relatively easy to accept the GM enzyme for cheese production, because the producing GM microbe is not present in the food production system, only the excreted chymosin, which is indistinguishable from the calf stomach version. In addition, the enzyme itself is also broken down during the cheese maturation process.

LAB have a long history of use by man, they are naturally present in raw food material and in the human gastro-intestinal tract. LAB are widely used as starter cultures for fermentation in the dairy, meat and other food industries. These food-grade bacteria can also improve the safety, shelf life, nutritional value, flavour and quality of the product. Moreover, LAB can be used as cell factories for the production of food additives and aroma compounds. It is further assumed that LAB may function as probiotics and contribute to the general health of the consumer. The uncontrolled genetic alterations of LAB that occur during random mutagenesis may lead to strains with altered properties. The level of such mutations depends on several environmental factors, e.g., radiation, mutagenic compounds and growth conditions. Selection of strains that have been subject- ed to uncontrolled genetic modifications is used to improve certain characteris- tics of the fermented end product, like flavour, structure, nutritional value. For

example, a random mutant Lactobacillus strain was isolated that was defective in lactate production, but had an increased level of the butter flavour compound diacetyl. LAB can also be selected for removal of undesirable compounds from raw food materials. In traditional yoghurt fermentation lactose is degraded only partially. One of the products is galactose, which may be harmful for people suffering from galactosemia. With laborious screening a galactose-fermenting spontaneous mutant Streptococcus strain was isolated, which solved this problem.

An alternative to random mutagenesis is targeted modification of the DNA. Genetic engineering offers a range of manipulations from single base pair substitutions, mutations, insertions of genes into the chromosome to deletion of portions DNA resulting in inactivation of specific enzymes. Food fermentation at large scale may suffer from bacteriophage infections resulting in lysis of the starter culture. The transformation of industrially important strains with phage resistance genes for other LAB could generate phage resistant strains. Another important feature of food products is texture. Gene clusters encoding exopolysaccharide producing enzymes have been transferred from one LAB strain to the other. The genetically modified strains improved the viscosity and texture of the fermented product.

As far as risk is concerned, it should be noted that despite the fact that the strains obtained via random mutagenesis are not considered GMOs, the occurrence of predictable unintended genetic alterations is very likely to take place within such mutant strains as well. The potential event of horizontal gene transfer following expression of foreign genes in LAB used in food fermentations is one of the major concerns that affect the safety assessment for consumer and environment. When the applied genetic elements originate from LAB that has a long and safe history of use in food, essentially no new risks are introduced.

13. Genetically modified plants as the basis of food